Fitzgerald M M, Trester M L, Jensen G M, McRee D E, Goodin D B
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Protein Sci. 1995 Sep;4(9):1844-50. doi: 10.1002/pro.5560040919.
The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.
细胞色素c过氧化物酶的活性状态，即化合物ES，在色氨酸-191侧链上含有一个阳离子自由基。我们最近报道，用甘氨酸取代该色氨酸会在活性位点形成一个埋藏腔，其中含有有序溶剂，并且会特异性结合质子化阳离子形式的取代咪唑（菲茨杰拉德MM，丘吉尔MJ，麦克里DE，古丁DB，1994年，《生物化学》33:3807 - 3818）。本研究探讨了附近的羧酸盐（天冬氨酸-235）和竞争性单价阳离子应调节W191G腔对配体结合亲和力的提议。咪唑鎓离子与W191G的竞争性结合滴定作为[K⁺]的函数表明，钾与咪唑的结合竞争较弱。在没有竞争性阳离子的情况下，观察到的钾结合解离常数（18 mM）比1,2 - 二甲基咪唑（5.5 μM）的解离常数高3000倍以上。值得注意的是，W191G - D235N双突变体没有显示出结合阳离子或中性形式咪唑的证据，尽管腔的结构在很大程度上不受羧酸盐取代的干扰。溶剂位置的精制晶体学B值表明，W191G中弱结合的钾在双突变体中显著减少。这些结果表明，天冬氨酸-235的埋藏负电荷是阳离子结合决定因素的一个基本特征，并表明该羧酸盐在稳定色氨酸-191自由基阳离子的形成中起关键作用。
